QC
Angie Chen
2024-01-30
Load libraries and working directory
Dimer check - Use sample_coverage files
## # A tibble: 15 × 2
## run dimer_percent
## <chr> <dbl>
## 1 M03 38.7
## 2 M04 22.4
## 3 M05 33.0
## 4 M06 60.7
## 5 M07 29.7
## 6 M08 24.5
## 7 M09 23.5
## 8 M10 25.0
## 9 M11 23.5
## 10 M12 26.3
## 11 M13 19.7
## 12 M14 11.3
## 13 M15 47.8
## 14 NS1 12.4
## 15 NS2 45.8Preview data structure
Merge allele, gene (amplicon info), and meta data
Preview raw data - sample size, loci numbers, allele numbers
## [1] "Sample size= 1630"## [1] "All locus = 274"## [1] "1 to 10 alleles per locus per sample"## [1] "Mean of total loci counts = 219.46, Mean prop. of monoclonal loci = 0.97, Mean prop. of polyclonal loci = 0.03"Preview raw data - Plate map, Detect contamination and Check
balanced
Check negative controls
## # A tibble: 17 × 1
## sampleID
## <chr>
## 1 M09_NC3
## 2 M13_NC3
## 3 M13_NC4
## 4 M13_NC7
## 5 M13_NC8
## 6 M15_NC3
## 7 M15_NC6
## 8 M13_NC1
## 9 M13_NC2
## 10 M13_NC6
## 11 M10_NC7
## 12 M13_NC5
## 13 M15_NC1
## 14 M15_NC4
## 15 M15_NC2
## 16 M07_NC-D05
## 17 M15_NC5
Check positive controls
## # A tibble: 32 × 2
## sampleID pc_dup_prop
## <chr> <dbl>
## 1 M07_PC-G04 0.867
## 2 M08_PC1 0.992
## 3 M08_PC2 0.996
## 4 M09_PC1 0.996
## 5 M09_PC2 0.987
## 6 M10_PC1 0.996
## 7 M10_PC2 0.987
## 8 M10_PC3 0.996
## 9 M11_PC1 0.987
## 10 M11_PC2 0.987
## # ℹ 22 more rowsRemove contaminated runs
## [1] "Sample size= 1239 Retained prop. = 0.76"## [1] "All locus = 274"## [1] "1 to 6 alleles per locus per sample"Data QC and filtering
- Filter out bad-quality samples and possible false positive alleles (off-targets, low-abundance)
- Make moire input file
Step 1. Total reads
## [1] "Step 1 - Sample size after removing samples with insufficient reads = 1043, Retained prop. = 0.84"## [1] "MH-05-AC_HAM14-000168" "MH-13-AC_HT29052011-3" "M14_HAM14-066-D0"
## [4] "M14_HT07032012-05" "MH-02-AC_HAM15-000700" "MH-02-AC_HAM15-000780"
## [7] "MH-02-AC_HAM15-000669" "MH-02-AC_HAM15-000734" "MH-02-AC_HAM15-000712"
## [10] "M14_HT20022011-01" "MH-02-AC_HAM15-000696" "MH-03-AC_HAM15-000636"
## [13] "MH-03-AC_HAM15-000750" "M14_HT05012011-03" "M14_HT11022013-07"
## [16] "M05_HAM16-185-D0" "MH-03-AC_HAM15-000210" "M14_HAM15-037-D0"
## [19] "M05_HAM16-169-D0" "M05_HAM16-111-D0" "M14_HT14122012-11"
## [22] "M05_HAM16-303-D0" "M05_HAM16-373-D0" "M12_HT23012013-01"
## [25] "M05_HAM16-182-D0" "MH-03-AC_HAM15-000561" "MH-11-AC_HT29072010-1"
## [28] "M05_HAM16-157-D0" "M05_HAM16-358-D0" "MH-02-AC_HAM15-000707"
## [31] "M12_HT19112012-01" "M14_HT11012013-07" "M12_HT17072012-07"
## [34] "M12_HT14122012-11" "M12_HT11012013-05" "MH-13-AC_HT08042011-3"
## [37] "MH-05-AC_HT02112013-1" "M12_HT22122012-07" "M12_HT14012013-05"
## [40] "MH-13-AC_HT13032011-4" "M12_HT02122012-01" "M12_HT11012013-03"
## [43] "M14_HT28062010-01" "MH-11-AC_HT07062010-2" "MH-12-AC_HT15022011-1"
## [46] "MH-03-AC_2007-15" "MH-03-AC_2007-3a" "MH-05-AC_HAM14-000163"
## [49] "MH-03-AC_2007-5a" "MH-03-AC_2007-7a" "MH-13-AC_HT09122010-2"
## [52] "MH-03-AC_2007-32" "MH-03-AC_2007-1a" "M03_HAM16-123-D0"
## [55] "M12_HT02022012-02" "MH-05-AC_HT01062013-7" "MH-13-AC_HT06112010-2"
## [58] "M12_HT21092012-03" "MH-11-AC_HT12042010-5" "MH-13-AC_HT18052011-21"
## [61] "MH-05-AC_HT27112013-6" "MH-05-AC_HT12072013-4" "MH-13-AC_HT23052011-2"
## [64] "MH-13-AC_HT28022011-1" "MH-11-AC_HT17062010-4" "MH-05-AC_HT03052013-11"
## [67] "MH-03-AC_2005-121" "MH-12-AC_HT10072010-1" "MH-12-AC_HT15042011-5"
## [70] "M03_HT11012013-03" "MH-09-AC_HT19112012-4" "MH-04-AC_HAM14-000378"
## [73] "M14_HAM14-128-D0" "M08_HAM15-266-D0" "MH-05-AC_HT02072013-14"
## [76] "M12_HT07072012-01" "MH-13-AC_HT28042011-1" "MH-04-AC_HAM14-000517"
## [79] "MH-12-AC_HT14062010-3" "M14_HT20072012-03" "M12_HT20072012-03"
## [82] "MH-04-AC_HAM14-000337" "MH-12-AC_HT06082010-1" "MH-06-AC_HT01072013-8"
## [85] "M14_HAM14-052-D0" "MH-06-AC_HT09022013-1" "M14_HAM14-141-D0"
## [88] "M14_HAM15-302-D0" "M08_HAM16-110-D0" "MH-12-AC_HT23072010-2"
## [91] "M14_HAM16-199-D0" "MH-05-AC_HAM14-000581" "M12_HAM16-199-D0"
## [94] "M14_HT14012013-05" "MH-05-AC_HAM14-000459" "MH-12-AC_HT18012011-2"
## [97] "M06_HAM16-260-D0" "MH-05-AC_HT31052013-6" "MH-12-AC_HT14122010-5"
## [100] "MH-12-AC_HT27122010-3" "MH-12-AC_HT04032011-2" "MH-04-AC_HAM14-000514"
## [103] "MH-13-AC_HT12122011-4" "M08_HAM15-122-D0" "MH-12-AC_HT20022011-2"
## [106] "M14_HAM14-108-D0" "MH-08-AC_HT13112012-5" "MH-05-AC_HAM14-000392"
## [109] "MH-12-AC_HT15112010-1" "M12_HT21072012-02" "MH-05-AC_HAM14-000233"Step 2. Loci coverage
## [1] "Step 2 - Sample size after removing samples with insufficeint loci coverage = 1022, Retained prop. = 0.98"## [1] "M11_HT16032010-05" "MH-04-AC_HAM14-000464" "MH-11-AC_HT09052010-3"
## [4] "MH-11-AC_HT26052010-1" "MH-12-AC_HT02112010-1" "MH-12-AC_HT02112010-2"
## [7] "MH-12-AC_HT05082010-1" "MH-12-AC_HT08052011-2" "MH-12-AC_HT13072010-1"
## [10] "MH-12-AC_HT15072010-2" "MH-12-AC_HT15122010-1" "MH-12-AC_HT16032011-1"
## [13] "MH-12-AC_HT16122010-1" "MH-12-AC_HT19112010-1" "MH-12-AC_HT20092010-2"
## [16] "MH-12-AC_HT20102010-3" "MH-12-AC_HT21052010-2" "MH-12-AC_HT24112010-1"
## [19] "MH-12-AC_HT27042011-3" "MH-13-AC_HT25072011-1" "MH-13-AC_HT28022011-5"Step 3: Proportion of loci > 100 reads in samples
## [1] "Step 3 - Sample size after removing samples with less than n100 cutoff = 992, Retained prop. = 0.97"## [1] "M06_HAM16-016-D0" "M06_HAM16-290-D0" "M08_HAM16-111-D0"
## [4] "M12_HAM15-290-D0" "M14_HAM15-623-D0" "MH-02-AC_HAM15-000628"
## [7] "MH-02-AC_HAM15-000754" "MH-05-AC_HT05102013-2" "MH-05-AC_HT13072013-3"
## [10] "MH-05-AC_HT15052013-1" "MH-05-AC_HT23062013-1" "MH-06-AC_HT24042013-5"
## [13] "MH-06-AC_HT29072013-1" "MH-06-AC_HT29092013-2" "MH-08-AC_HT03082012-4"
## [16] "MH-08-AC_HT04062012-6" "MH-08-AC_HT04122012-12" "MH-08-AC_HT14072012-1"
## [19] "MH-08-AC_HT16062012-11" "MH-08-AC_HT17112012-4" "MH-08-AC_HT22122012-3"
## [22] "MH-08-AC_HT24082012-2" "MH-12-AC_HT11112010-1" "MH-12-AC_HT14122010-7"
## [25] "MH-12-AC_HT18042011-8" "MH-12-AC_HT29122010-1" "MH-13-AC_HT05022011-2"
## [28] "MH-13-AC_HT08032011-5" "MH-13-AC_HT23032012-4" "MH-13-AC_HT29042011-8"Check if there are any loci not amplified well, and if this happened in one sample or many samples
## [1] "126 loci had <= 10 reads at least in one sample"## [1] "2 loci had <= 10 reads across 1/3 of total samples"## [1] "Which loci had <= 10 reads across 1/3 of total samples?"## [1] "Pf3D7_03_v3-240957-241256-2" "Pf3D7_09_v3-685560-685825-1A"qPCR density and sample info
Optional - remove minor alleles
## [1] "Percentage (x100) of excluded minor alleles in total alleles = 3.088, Percentage (x100) of excluded minor alleles in total data entries = 0.078 (x100)"## [1] "Percentage (x100) of excluded minor alleles in total alleles = 3.088, Percentage (x100) of excluded minor alleles in total data entries = 0.078 (x100)"## [1] "t10.108" "t10.109" "t10.128" "t10.13" "t10.150" "t10.155"
## [7] "t10.164" "t10.25" "t10.29" "t10.65" "t10.67" "t10.70"
## [13] "t10.73" "t10.76" "t108.3" "t111.19" "t140.11" "t140.12"
## [19] "t140.13" "t140.16" "t140.48" "t140.53" "t140.55" "t140.58"
## [25] "t141.27" "t154.17" "t154.49" "t154.52" "t154.6" "t155.125"
## [31] "t155.66" "t156.17" "t156.43" "t156.65" "t16.11" "t162.27"
## [37] "t164.16" "t167.19" "t17.55" "t17.59" "t17.68" "t171.39"
## [43] "t171.48" "t181.10" "t19.4" "t192.36" "t193.103" "t193.16"
## [49] "t193.29" "t193.3" "t193.31" "t193.44" "t194.12" "t194.20"
## [55] "t194.43" "t194.44" "t194.9" "t196.26" "t2.24" "t200.19"
## [61] "t207.23" "t207.25" "t207.30" "t21.2" "t21.33" "t217.3"
## [67] "t217.33" "t217.35" "t219.52" "t223.3" "t223.50" "t223.66"
## [73] "t231.18" "t231.4" "t239.8" "t245.5" "t246.23" "t250.8"
## [79] "t258.18" "t262.39" "t266.19" "t266.43" "t266.49" "t266.53"
## [85] "t272.35" "t4.14" "t4.16" "t4.24" "t4.41" "t4.44"
## [91] "t4.45" "t4.47" "t4.48" "t4.49" "t4.50" "t4.52"
## [97] "t4.55" "t4.56" "t4.58" "t4.61" "t40.18" "t40.21"
## [103] "t49.21" "t49.3" "t6.39" "t6.40" "t60.28" "t85.3"
## [109] "t90.20"Make moire input
Moire input